Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer--Childhood Leukemia Cooperative Group.

BACKGROUND AND METHODS: The implications of the detection of residual disease after treatment of acute lymphoblastic leukemia (ALL) are unclear. We conducted a prospective study at 11 centers to determine the predictive value of the presence or absence of detectable residual disease at several points in time during the first six months after complete remission of childhood ALL had been induced. Junctional sequences of T-cell-receptor or immunoglobulin gene rearrangements were used as clonal markers of leukemic cells. Residual disease was quantitated with a competitive polymerase-chain-reaction (PCR) assay. Of 246 patients enrolled at diagnosis and treated with a uniform chemotherapy protocol, 178 were monitored for residual disease with one clone-specific probe (in 74 percent) or more than one probe (in 26 percent). The median follow-up period was 38 months. RESULTS: The presence or absence and level of residual leukemia were significantly correlated with the risk of early relapse at each of the times studied (P<0.001). PCR measurements identified patients at high risk for relapse after the completion of induction therapy (those with > or =10(-2) residual blasts) or at later time points (those with > or =10(-3) residual blasts). Multivariate analysis showed that as compared with immunophenotype, age, risk group (standard or very high risk), and white-cell count at diagnosis, the presence or absence and level of residual disease were the most powerful independent prognostic factors. CONCLUSIONS: Residual leukemia after induction of a remission is a powerful prognostic factor in childhood ALL. Detection of residual disease by PCR should be used to identify patients at risk for relapse and should be taken into account in considering alternative treatment.

Expansion of polyclonal B-cell precursors in bone marrow from children treated for acute lymphoblastic leukemia.

In a series of 12 patients (mean age: 3 years at diagnosis) receiving chemotherapy for acute lymphoblastic leukemia, bone marrow examinations performed during hematopoietic recovery following treatment-induced agranulocytosis or completion of maintenance treatment showed at least 15% of non malignant immature cells which were sometimes hardly distinguishable from leukemic cells. No comparable data was observed in patients treated with G-CSF. The cytological features of these cells as well as their immunophenotyping were defined. Results showed that the majority of cells expressed HLA-DR, CD19, CD10 and cytoplasmic IgM but not the CD34 markers. This predominant and homogeneous pre-B cell population which likely represents the expansion of a minor population detectable in normal bone marrow is phenotypically indistinguishable from leukemic cells. The pattern of IgH gene rearrangements studied by PCR amplification of the CDRIII region showed that these cells were polyclonal. Except in one patient, minimal residual disease was not detected using probes specific for IgH or TCR gene rearrangement of the malignant clone. In children during the hematopoietic recovery after chemotherapy, immature marrow cells in great numbers, even with an highly homogeneous immunophenotype identical to the malignant clone's, are not sufficient for the diagnosis of relapse.

Delineation of a 6 cM commonly deleted region in childhood acute lymphoblastic leukemia on the 6q chromosomal arm.

Deletion of the long arm of human chromosome 6 in acute lymphoblastic leukemia (ALL) has been shown by cytogenetic studies in 4-11% of cases. To characterize further the region of deletion and to precisely establish its frequency, we studied loss of heterozygosity (LOH) in 120 children with ALL using polymorphic markers located from the 6q14-15 chromosomal band to the telomere. LOH was detected in eight patients. A single region of LOH, flanked distally by D6S1594 and proximally by D6S301 was detected. These DNA markers are separated by 6 cM and are approximately located at the 6q21-22 band. Our present results delineate a region that is likely to contain a tumor-suppressor gene involved in a subset of childhood ALLs.

A low rate of loss of heterozygosity is found at many different loci in childhood B-lineage acute lymphocytic leukemia.

We have carried out a screening for loss of heterozygosity (LOH) in 51 children with B-lineage acute lymphoblastic leukemia (ALL). Forty-six highly polymorphic microsatellite markers located in subtelomeric areas of every chromosome arm were analyzed in each patient. Allelic losses were encountered at 21 of the 46 loci tested (46%). The frequency of LOH at a given locus was usually < 10% and fractional allelic loss, calculated as the ratio of chromosomal arms displaying loss among all informative arms for each patient, ranged from 0.025 to 0.31 (mean, 0.063). This study provides further evidence that deletional events are a major type of genetic alteration found in childhood ALL. The diversity of the loci displaying LOH suggests that, as in solid tumors, numerous tumor suppressor genes are likely to participate in leukemogenesis. However, the overall low frequency of LOH, as well as the absence of microsatellite instability suggest that the genetic instability is lower in childhood ALL than in most of the solid tumors.

Loss of heterozygosity in the chromosomal region 12p12-13 is very common in childhood acute lymphoblastic leukemia and permits the precise localization of a tumor-suppressor gene distinct from p27KIP1.

Abnormalities of the short arm of chromosome 12 are relatively common in hematologic malignancies and deletions of the region. 12p12-13 are found in approximately 5% of the patients with acute lymphoblastic leukemia (ALL). As a potent inhibitor of cyclin-dependent kinases, p27KIP1 prevents the progression of the cell cycle and the gene encoding p27KIP1 represents a potential tumor-suppressor gene. Its recent assignment to the chromosomal region (12p12.3) prompted us to study the p27KIP1 gene in a series of 61 children with ALL. Microsatellite polymorphic markers flanking the p27KIP1 gene were analyzed to detect losses of heterozygosity (LOH). Eleven patients displayed LOH for at least one of the markers. The deleted are encompassed the p27KIP1 gene locus in 10 cases, but inactivation of the remaining allele by deletion, translocation, or mutation was never observed. In addition, in 1 patient, the p27KIP1 gene was situated outside of the region of LOH. Thus, p27KIP1 does not seem to be the target gene of 12p12-13 alterations. However, this study indicates that 12p12-13 alterations at the molecular level, which are present in about 27% of the children with B-lineage ALL, are much more common than had previously been reported by usual chromosome analysis. Moreover, LOH mapping allowed us to better define the location of a putative tumor-suppressor gene implicated in these malignancies and should therefore help in identifying this gene.

Potential antileukemic effect of gamma delta T cells in acute lymphoblastic leukemia.

The immune response to leukemia is poorly understood. We postulated that nonmalignant T lymphocytes remaining within bone marrow from children with newly diagnosed ALL could be involved in this immune response. T lymphocytes which expressed gamma delta TCR comprised less than 1% of ALL marrow cells. A preferential outgrowth of gamma delta T cells within the CD3 population was observed when marrow cells were cultured with IL-2 alone or with stimulating feeder cells. These results, obtained in a series of 14 patients with precursor B-ALL, were significantly different when compared with expansions from normal marrow cells. In one patient, the clones established from the expanded population displayed different patterns of cytotoxicity against tumoral targets of the B cell lineage. Some clones expressing the TCR V delta 1 segment showed cytotoxic activity against a cell line derived from a pre-B ALL without activity against a LAK-sensitive B cell line. Using PCR amplification, one such clone was detected at high frequency, in the primary expansion of ALL marrow cells. These results suggest a prior activation in vivo of some gamma delta T cells by leukemic cells and provide some evidence on the role of these subsets in the immune response to leukemia.

Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes.

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B-lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

Sequence correction and reassignment of the TaqI polymorphic site in the human inter-gamma-globin gene region, an African-specific marker.

We report here that the assignment of nt 37504 in the human inter-gamma-globin gene region of the HUMHBB locus sequence as a C is incorrect and should be replaced by a T. Accordingly, the polymorphic TaqI site, originally described at position 37503 as an African-specific marker, is actually located at position 37992. A PCR-based assay for this anthropologically important genetic marker is described.

Analysis of the 5' flanking sequence of the G gamma globin gene by denaturing gradient gel electrophoresis confirms the heterogeneity of the Bantu beta S haplotype.

The GC-->TT polymorphism recently described at positions -1106 and -1105 in the 5' flanking region of the G gamma globin gene for the Bantu beta S haplotype was analysed by denaturing gradient gel electrophoresis. We studied 108 beta S chromosome and 122 beta A chromosomes. The TT sequence was found as follows: in all of 80 chromosomes bearing the Bantu beta S haplotype with the 6-bp deletion -400 nt from the G gamma gene in 3 out of 5 Bantu beta S chromosomes without the deletion, in 1 out of 122 beta A chromosomes from different ethnic origins but in none of 23 beta S chromosomes bearing the Senegal, Benin or Cameroon haplotypes. These results confirm the heterogeneity of the Bantu beta S haplotype and allow a tentative evolutionary sequence for the different alleles at this locus to be presented.